Tobacco consumption, driven by addiction to nicotine, is the leading cause of preventable death in the United States. In the period from 1995-1999, the CDC reported 440,00 deaths/year from illnesses attributable to smoking (cancer, respiratory, and cardiovascular diseases) and $157 billion/year in related economic losses. Neuronal nicotinic acetylcholine receptors (nAChRs) initiate and reinforce the process of nicotine addiction. These receptor molecules aggregate at pre- and postsynaptic sites in the brain and autonomic nervous system, allowing them to both modulate and directly mediate synaptic transmission once activated by acetylcholine (ACh) released from cholinergic nerve terminals. Recent studies demonstrate that chronic exposure to levels of nicotine seen in smokers' blood, causes sustained changes in both pre- and postsynaptic nAChRs. It is widely presumed that such changes alter synaptic circuits underlying addictive behaviors associated with nicotine, and possibly other commonly abused drugs. Unfortunately, these circuits are embedded in brain regions where experimental control of pre- and postsynaptic elements is difficult or impossible to achieve, preventing discovery of precise mechanisms relevant to nicotine-induced changes in synaptic function. To address this problem, our laboratory proposes to combine state of the art single-cell gene transfection and high-resolution synaptic stimulation/recording methods in a model culture system where nAChRs and nicotinic synapses are abundant and accessible. One goal is to manipulate gene expression in pre- and postsynaptic neurons that can subsequently be examined in detailed electrophysiological experiments (Aim 1). Realizing this goal will provide a cutting-edge advance relevant to many studies of synapse formation, function and regulation. A second goal is to test the utility of our approach by probing for alterations in synaptic function expected to accompany sustained changes in nicotinic receptor activation (Aim 2). It is anticipated that these latter experiments will provide new answers concerning how alterations of nicotinic synaptic components modify circuit function to initiate and reinforce addiction to nicotine and other abused drugs. [unreadable] [unreadable] [unreadable]